Cartilage tissues regulate systemic aging via ectonucleotide pyrophosphatase/phosphodiesterase 1 in mice.

Aging presents fundamental health concerns worldwide; however, mechanisms underlying how aging is regulated are not fully understood. Here, we show that cartilage regulates aging by controlling phosphate metabolism via ectonucleotide pyrophosphatase/phosphodiesterase 1 (Enpp1). We newly established an Enpp1 reporter mouse, in which an EGFP-luciferase sequence was knocked-in at the Enpp1 gene start codon (Enpp1/EGFP-luciferase), enabling detection of Enpp1 expression in cartilage tissues of resultant mice. We then established a cartilage-specific Enpp1 conditional knockout mouse (Enpp1 cKO) by generating Enpp1 flox mice and crossing them with cartilage-specific type 2 collagen Cre mice. Relative to WT controls, Enpp1 cKO mice exhibited phenotypes resembling human aging, such as short life span, ectopic calcifications, and osteoporosis, as well as significantly lower serum pyrophosphate levels. We also observed significant weight loss and worsening of osteoporosis in Enpp1 cKO mice under phosphate overload conditions, similar to global Enpp1-deficient mice. Aging phenotypes seen in Enpp1 cKO mice under phosphate overload conditions were rescued by a low vitamin D diet, even under high phosphate conditions. These findings suggest overall that cartilage tissue plays an important role in regulating systemic aging via Enpp1.

The IL-17-IL-17RA axis is required to promote osteosarcoma progression in mice.

Osteosarcoma is rare but is the most common bone tumor. Diagnostic tools such as magnetic resonance imaging development of chemotherapeutic agents have increased the survival rate in osteosarcoma patients, although 5-year survival has plateaued at 70%. Thus, development of new treatment approaches is needed. Here, we report that IL-17, a proinflammatory cytokine, increases osteosarcoma mortality in a mouse model with AX osteosarcoma cells. AX cell transplantation into wild-type mice resulted in 100% mortality due to ectopic ossification and multi-organ metastasis. However, AX cell transplantation into IL-17-deficient mice significantly prolonged survival relative to controls. CD4-positive cells adjacent to osteosarcoma cells express IL-17, while osteosarcoma cells express the IL-17 receptor IL-17RA. Although AX cells can undergo osteoblast differentiation, as can patient osteosarcoma cells, IL-17 significantly inhibited that differentiation, indicating that IL-17 maintains AX cells in the undifferentiated state seen in malignant tumors. By contrast, IL-17RA-deficient mice transplanted with AX cells showed survival comparable to wild-type mice transplanted with AX cells. Biopsy specimens collected from osteosarcoma patients showed higher expression of IL-17RA compared to IL-17. These findings suggest that IL-17 is essential to maintain osteosarcoma cells in an undifferentiated state and could be a therapeutic target for suppressing tumorigenesis.

Transthyretin amyloid deposition in ligamentum flavum (LF) is significantly correlated with LF and epidural fat hypertrophy in patients with lumbar spinal stenosis.

Lumbar spinal stenosis (LSS) is a degenerative disease characterized by intermittent claudication and numbness in the lower extremities. These symptoms are caused by the compression of nerve tissue in the lumbar spinal canal. Ligamentum flavum (LF) hypertrophy and spinal epidural lipomatosis in the spinal canal are known to contribute to stenosis of the spinal canal: however, detailed mechanisms underlying LSS are still not fully understood. Here, we show that surgically harvested LFs from LSS patients exhibited significantly increased thickness when transthyretin (TTR), the protein responsible for amyloidosis, was deposited in LFs, compared to those without TTR deposition. Multiple regression analysis, which considered age and BMI, revealed a significant association between LF hypertrophy and TTR deposition in LFs. Moreover, TTR deposition in LF was also significantly correlated with epidural fat (EF) thickness based on multiple regression analyses. Mesenchymal cell differentiation into adipocytes was significantly stimulated by TTR in vitro. These results suggest that TTR deposition in LFs is significantly associated with increased LF hypertrophy and EF thickness, and that TTR promotes adipogenesis of mesenchymal cells. Therapeutic agents to prevent TTR deposition in tissues are currently available or under development, and targeting TTR could be a potential therapeutic approach to inhibit LSS development and progression.

Remnant tissue enhances early postoperative biomechanical strength and infiltration of Scleraxis-positive cells within the grafted tendon in a rat anterior cruciate ligament reconstruction model.

When ruptured, ligaments and tendons have limited self-repair capacity and rarely heal spontaneously. In the knee, the Anterior Cruciate Ligament (ACL) often ruptures during sports activities, causing functional impairment and requiring surgery using tendon grafts. Patients with insufficient time to recover before resuming sports risk re-injury. To develop more effective treatment, it is necessary to define mechanisms underlying ligament repair. For this, animal models can be useful, but mice are too small to create an ACL reconstruction model. Thus, we developed a transgenic rat model using control elements of Scleraxis (Scx), a transcription factor essential for ligament and tendon development, to drive GFP expression in order to localize Scx-expressing cells. As anticipated, Tg rats exhibited Scx-GFP in ACL during developmental but not adult stages. Interestingly, when we transplanted the flexor digitorum longus (FDP) tendon derived from adult Scx-GFP+ rats into WT adults, Scx-GFP was not expressed in transplanted tendons. However, tendons transplanted from adult WT rats into Scx-GFP rats showed upregulated Scx expression in tendon, suggesting that Scx-GFP+ cells are mobilized from tissues outside the tendon. Importantly, at 4 weeks post-surgery, Scx-GFP-expressing cells were more frequent within the grafted tendon when an ACL remnant was preserved (P group) relative to when it was not (R group) (P vs R groups (both n = 5), p<0.05), and by 6 weeks, biomechanical strength of the transplanted tendon was significantly increased if the remnant was preserved (P vsR groups (both n = 14), p<0.05). Scx-GFP+ cells increased in remnant tissue after surgery, suggesting remnant tissue is a source of Scx+ cells in grafted tendons. We conclude that the novel Scx-GFP Tg rat is useful to monitor emergence of Scx-positive cells, which likely contribute to increased graft strength after ACL reconstruction.

A machine learning-based scoring system and ten factors associated with hip fracture occurrence in the elderly.

Hip fractures are fragility fractures frequently seen in persons over 80-years-old. Although various factors, including decreased bone mineral density and a history of falls, are reported as hip fracture risks, few large-scale studies have confirmed their relevance to individuals older than 80, and tools to assess contributions of various risks to fracture development and the degree of risk are lacking. We recruited 1395 fresh hip fracture patients and 1075 controls without hip fractures and comprehensively evaluated various reported risk factors and their association with hip fracture development. We initially constructed a predictive model using Extreme Gradient Boosting (XGBoost), a machine learning algorithm, incorporating all 40 variables and evaluated the model's performance using the area under the receiver operating characteristic curve (AUC), yielding a value of 0.87. We also employed SHapley Additive exPlanation (SHAP) values to evaluate each feature importance and ranked the top 20. We then used a stepwise selection method to determine key factors sequentially until the AUC reached a plateau nearly equal to that of all variables and identified the top 10 sufficient to evaluate hip fracture risk. For each, we determined the cutoff value for hip fracture occurrence and calculated scores of each variable based on the respective feature importance. Individual scores were: serum 25(OH)D levels (<10 ng/ml, score 7), femoral neck T-score (<-3, score 5), Barthel index score (<100, score 3), maximal handgrip strength (<18 kg, score 3), GLFS-25 score (≥24, score 2), number of falls in previous 12 months (≥3, score 2), serum IGF-1 levels (<50 ng/ml, score 2), cups of tea/day (≥5, score -2), use of anti-osteoporosis drugs (yes, score -2), and BMI (<18.5 kg/m2, score 1). Using these scores, we performed receiver operating characteristic (ROC) analysis and the resultant optimal cutoff value was 7, with a specificity of 0.78, sensitivity of 0.75, and AUC of 0.85. These ten factors and the scoring system may represent tools useful to predict hip fracture.

Potential function of Scx+/Sox9+ cells as progenitor cells in rotator cuff tear repair in rats.

Tendons and their attachment sites to bone, fibrocartilaginous tissues, have poor self-repair capacity when they rupture, and have risks of retear even after surgical repair. Thus, defining mechanisms underlying their repair is required in order to stimulate tendon repairing capacity. Here we used a rat surgical rotator cuff tear repair model and identified cells expressing the transcription factors Scleraxis (Scx) and SRY-box 9 (Sox9) as playing a crucial role in rotator cuff tendon-to-bone repair. Given the challenges of establishing stably reproducible models of surgical rotator cuff tear repair in mice, we newly established Scx-GFP transgenic rats in which Scx expression can be monitored by GFP. We observed tissue-specific GFP expression along tendons in developing ScxGFP transgenic rats and were able to successfully monitor tissue-specific Scx expression based on GFP signals. Among 3-, 6-, and 12-week-old ScxGFP rats, Scx+/Sox9+ cells were most abundant in 3-week-old rats near the site of humerus bone attachment to the rotator cuff tendon, while we observed significantly fewer cells in the same area in 6- or 12-week-old rats. We then applied a rotator cuff repair model using ScxGFP rats and observed the largest number of Scx+/Sox9+ cells at postoperative repair sites of 3-week-old relative to 6- or 12-week-old rats. Tendons attach to bone via fibrocartilaginous tissue, and cartilage-like tissue was seen at repair sites of 3-week-old but not 6- or 12-week-old rats during postoperative evaluation. Our findings suggest that Scx+/Sox9+ cells may function in rotator cuff repair, and that ScxGFP rats could serve as useful tools to develop therapies to promote rotator cuff repair by enabling analysis of these activities.

[Accuracy of Virtual Non-contrast Image Reconstruction Using Material Decomposition for Fast kV-switching Dual-energy CT].

PURPOSE: Dual-energy computed tomography (DECT) system can generate virtual non-contrast (VNC) images. Although several reconstruction algorithms are defined, there are not many researches using deep learning image reconstruction (DLIR) algorithm. In this study, we evaluated the accuracy of the VNC image reconstruction under various conditions using DLIR algorithm. METHODS: At first, each iodine insert with variable concentrations (2.0, 5.0, 10.0, 15.0 mg/ml) or diameters (2.0, 5.0, 10.0, 28.5 mm), or mixed insert including blood-mimicking material with iodine (iodine concentrations: 2.0, 4.0 mg/ml) was put in the center of the multi-energy CT phantom (Gammex, USA). This phantom was placed in the isocenter of DECT, and it scanned and reconstructed the VNC images. In addition, the VNC images were reconstructed with various display field of view (DFOV) sizes (240, 350 mm) or reconstruction algorithms (filtered back projection, advanced statistical iterative reconstruction, deep learning image reconstruction) for each iodine diameter. Attenuation values of these images (CTVNC) were measured and assessed by placing a circular region of interest (ROI) on each insert. RESULTS: CTVNC form iodine inserts increased with iodine concentration became lower, whereas CTVNC form blood plus iodine inserts were stable regardless of low iodine concentration. As iodine diameter became smaller, CTVNC increased remarkably. CTVNC remained steady even though reconstruction parameters were varied. CONCLUSION: In our study, the VNC image reconstruction using DLIR algorithm was affected by various conditions such as iodine concentration and size. In particular, its accuracy was reduced by the size of target.

Image Quality Assessment of Deep Learning Image Reconstruction in Torso Computed Tomography Using Tube Current Modulation.

Novel deep learning image reconstruction (DLIR) reportedly changes the image quality characteristics based on object contrast and image noise. In clinical practice, computed tomography image noise is usually controlled by tube current modulation (TCM) to accommodate changes in object size. This study aimed to evaluate the image quality characteristics of DLIR for different object sizes when the in-plane noise was controlled by TCM. Images acquisition was performed on a GE Revolution CT system to investigate the impact of the DLIR algorithm compared to the standard reconstructions of filtered-back projection (FBP) and hybrid iterative reconstruction (hybrid-IR). The image quality assessment was performed using phantom images, and an observer study was conducted using clinical cases. The image quality assessment confirmed the excellent noise- reduction performance of DLIR, despite variations due to phantom size. Similarly, in the observer study, DLIR received high evaluations regardless of the body parts imaged. We evaluated a novel DLIR algorithm by replicating clinical behaviors. Consequently, DLIR exhibited higher image quality than those of FBP and hybrid-IR in both phantom and observer studies, albeit the value depended on the reconstruction strength, and proved itself capable of providing stable image quality in clinical use.

Angiopoietin-Like Protein 2 Induces Synovial Inflammation in the Facet Joint Leading to Degenerative Changes via Interleukin-6 Secretion.

STUDY DESIGN: Experimental human study. PURPOSE: To determine whether angiopoietin-like protein 2 (ANGPTL2) is highly expressed in the hyperplastic facet joint (FJ) synovium and whether it activates interleukin-6 (IL-6) secretion in FJ synoviocytes. OVERVIEW OF LITERATURE: Mechanical stress-induced synovitis is partially, but significantly, responsible for degenerative and subsequently osteoarthritic changes in the FJ tissues in patients with lumbar spinal stenosis (LSS). However, the underlying molecular mechanism remains unclear. IL-6 is highly expressed in degenerative FJ synovial tissue and is responsible for local chronic inflammation. ANGPTL2, an inflammatory and mechanically induced mediator, promotes the expression of IL-6 in many cells. METHODS: FJ tissues were harvested from five patients who had undergone lumbar surgery. Immunohistochemistry for ANGPTL2, IL-6, and cell markers was performed in the FJ tissue samples. After cultured synoviocytes from the FJ tissues were subjected to mechanical stress, ANGPTL2 expression and secretion were measured quantitatively using real-time quantitative reverse-transcription-polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA), respectively. Following ANGPTL2 administration in the FJ synoviocytes, anti-nuclear factor-κB (NF-κB) activation was investigated using immunocytochemistry, and IL-6 expression and secretion were assayed quantitatively with or without NF-κB inhibitor. Moreover, we assessed whether ANGPTL2-induced IL-6 modulates leucocyte recruitment in the degenerative process by focusing on the monocyte chemoattractant protein-1 (MCP-1) expression. RESULTS: ANGPTL2 and IL-6 were highly expressed in the hyperplastic FJ synovium samples. ANGPTL2 was co-expressed in both, fibroblast-like and macrophage-like synoviocytes. Further, the expression and secretion of ANGPTL2 in the FJ synoviocytes increased in response to stimulation by mechanical stretching. ANGPTL2 protein promoted the nuclear translocation of NF-κB and induced IL-6 expression and secretion in the FJ synoviocytes. This effect was reversed following treatment with NF-κB inhibitor. Furthermore, ANGPTL2-induced IL-6 upregulated the MCP-1 expression in the FJ synoviocytes. CONCLUSIONS: Mechanical stress-induced ANGPTL2 promotes chronic inflammation in the FJ synovium by activating IL-6 secretion, leading to FJ degeneration and subsequent LSS.

Matrix metalloproteinase promotes elastic fiber degradation in ligamentum flavum degeneration.

Ligamentum flavum (LF) hypertrophy in lumbar spinal canal stenosis (LSCS) is characterized by a loss of elastic fibers and fibrosis. Chronic inflammation is thought to be responsible for the histological change but the mechanism underlying elastic fiber degradation remains unclear. Given that matrix metalloproteinase (MMP)-2 and -9 have elastolytic activity and are partly regulated by inflammatory cytokines such as interleukin (IL)-6, in this study, we investigated whether MMPs mediate LF degeneration using 52 LF samples obtained during lumbar surgery, including 31 LSCS and 21 control specimens. We confirmed by histological analysis that the LSCS samples exhibited severe degenerative changes compared with the controls. We found that MMP-2 was upregulated in LF tissue from patients with LSCS at the mRNA and protein levels, whereas MMP-9 expression did not differ between the two groups. The MMP-2 level was positively correlated with LF thickness and negatively correlated with the area occupied by elastic fibers. IL-6 mRNA expression was also increased in LF tissue from patients with LSCS and positively correlated with that of MMP-2. Signal transducer and activator of transcription (STAT)3, a component of the IL-6 signaling pathway, was activated in hypertrophied LF tissues. Our in vitro experiments using fibroblasts from LF tissue revealed that IL-6 increased MMP-2 expression, secretion, and activation via induction of STAT3 signaling, and this effect was reversed by STAT3 inhibitor treatment. Moreover, elastin degradation was promoted by IL-6 stimulation in LF fibroblast culture medium. These results indicate that MMP-2 induction by IL-6/STAT3 signaling in LF fibroblasts can degrade elastic fibers, leading to LF degeneration in LSCS.

Mesenchymal stem cells derived from human iPS cells via mesoderm and neuroepithelium have different features and therapeutic potentials.

Mesenchymal stem cells (MSCs) isolated from adult human tissues are capable of proliferating in vitro and maintaining their multipotency, making them attractive cell sources for regenerative medicine. However, the availability and capability of self-renewal under current preparation regimes are limited. Induced pluripotent stem cells (iPSCs) now offer an alternative, similar cell source to MSCs. Herein, we established new methods for differentiating hiPSCs into MSCs via mesoderm-like and neuroepithelium-like cells. Both derived MSC populations exhibited self-renewal and multipotency, as well as therapeutic potential in mouse models of skin wounds, pressure ulcers, and osteoarthritis. Interestingly, the therapeutic effects differ between the two types of MSCs in the disease models, suggesting that the therapeutic effect depends on the cell origin. Our results provide valuable basic insights for the clinical application of such cells.

Evaluation of the utility of the Estimation of Physiologic Ability and Surgical Stress score for predicting post-operative morbidity after orthopaedic surgery.

PURPOSE: The purpose of this study was to investigate the utility of the Estimation of Physiologic Ability and Surgical Stress (E-PASS) scoring system for predicting post-operative morbidity. METHODS: We included 1,883 patients (mean age, 52.1 years) who underwent orthopaedic surgery. The post-operative complications were classified as surgical site and non-surgical site complications, and the relationship between the E-PASS scores and post-operative morbidity was investigated. RESULTS: The incidence of post-operative complications (n = 274) significantly increased with an increase in E-PASS scores (p < 0.001). The areas under the curve for the comprehensive risk score of the E-PASS scoring system for overall and non-surgical site complications were 0.777 and 0.794, respectively. CONCLUSIONS: The E-PASS scoring system showed some utility in predicting post-operative morbidity after general orthopaedic surgery. However, creating a new risk score that is more suitable for orthopaedic surgery will be challenging.

Local Application of Gelatin Hydrogel Sheets Impregnated With Platelet-Derived Growth Factor BB Promotes Tendon-to-Bone Healing After Rotator Cuff Repair in Rats.

PURPOSE: To determine whether the local application of platelet-derived growth factor BB (PDGF-BB) in hydrogel sheets would promote healing and improve histologic characteristics and biomechanical strength after rotator cuff (RC) repair in rats. METHODS: To assess the effect of PDGF-BB on tendon-to-bone healing we divided 36 adult male Sprague-Dawley rats treated with bilateral surgery to repair the supraspinatus tendon at its insertion site into 3 groups: group 1 = suture-only group; group 2 = suture and gelatin hydrogel sheets impregnated with phosphate-buffered saline (PBS); and group 3 = suture and gelatin hydrogel sheets impregnated with PDGF-BB (0.5 µg). Semiquantitative histologic evaluation was carried out 2, 6, and 12 weeks later; cell proliferation was assessed 2 and 6 weeks postoperatively by immunostaining for proliferating cell nuclear antigen (PCNA), and biomechanical testing, including ultimate load to failure, stiffness, and ultimate stress to failure, was performed 12 weeks after the operation. RESULTS: At 2 weeks, the average percentage of PCNA-positive cells at the insertion site was significantly higher in group 3 (40.5% ± 2.4%) than in group 1 (32.1% ± 6.9%; P = .03) and group 2 (31.9% ± 3.7%; P = .02). At 2 and 6 weeks, the histologic scores were similar among the 3 groups. At 12 weeks, the histologic score was significantly higher in group 3 (10.3 ± 0.8) than in group 1 (8.5 ± 0.5; P = .002) or group 2 (8.8 ± 0.8; P = .009), whereas ultimate load to failure, stiffness, and ultimate load to stress (normal control population, 44.73 ± 9.75 N, 27.59 ± 4.32 N/mm, and 21.33 ± 4.65 N/mm(2), respectively) were significantly higher in group 3 (28.28 ± 6.28 N, 11.05 ± 2.37 N/mm, and 7.99 ± 2.13 N/mm(2), respectively) than in group 1 (10.44 ± 1.98 N, 4.74 ± 1.31 N/mm, and 3.28 ± 1.27 N/mm(2), respectively; all P < .001) or group 2 (11.85 ± 2.89 N, 5.86 ± 1.75 N/mm, and 3.31 ± 0.80 N/mm(2), respectively; all P < .001). CONCLUSIONS: The placement of a PDGF-BB-impregnated hydrogel sheet just lateral to a transected and acutely reattached supraspinatus tendon produced significantly more PCNA-positive cells at 2 weeks and greater collagen fiber orientation, ultimate failure loads, stiffness, and stress to failure at 12 weeks than did a PBS-impregnated hydrogel sheet. No differences in vascularity or cellularity were observed. CLINICAL RELEVANCE: The local application of PDGF-BB-impregnated gelatin hydrogel may help to promote tendon-to-bone healing after RC repair in humans.

Intracellular accumulation of advanced glycation end products induces apoptosis via endoplasmic reticulum stress in chondrocytes.

Mammalian cells attempt to maintain their homeostasis under endoplasmic reticulum (ER) stress. If the stress cannot be alleviated, cells are led to apoptosis through induction of C/EBP homologous protein (CHOP). ER stress is provoked in osteoarthritis chondrocytes, and intracellular accumulation of advanced glycation end products (AGEs) in chondrocytes is a possible cause. To clarify the role of intracellular AGE accumulation in chondrocytes, the present study investigated the effect of intracellular AGE accumulation on ER stress and apoptosis by in vitro and in vivo analysis. Intracellular AGE accumulation induced by AGE precursors caused apoptosis, induced expression of ER stress markers, and led to co-localization of AGEs with glucose-regulated protein 78, leading to formation of high-molecular-weight complexes in cultured chondrocytes. These reactions were inhibited by an AGE formation inhibitor. CHOP deletion inhibited apoptosis induced by intracellular AGE accumulation. In vivo intracellular AGE accumulation induced by intra-articular injection of AGE precursors caused ER stress and apoptosis in chondrocytes and led to degradation of articular cartilage. Additionally, intracellular AGE accumulation increased the degree of cartilage degradation in an osteoarthritis model. These data indicate that intracellular accumulation of AGEs induces modification of unfolded protein response-related protein by AGEs and apoptosis via ER stress in chondrocytes. Moreover, the in vivo study showed that intracellular AGE accumulation in chondrocytes is involved in the occurrence and progression of osteoarthritis through ER stress. Thus, research on mechanisms of apoptosis via ER stress induced by intracellular AGE accumulation in chondrocytes will lead to a new understanding of osteoarthritis pathology.